Screening of Synergistic Interactions of Epigallocatechin-3-gallate with Antiangiogenic and Antitumor Compounds

Accepted Manuscript Title: Screening of Synergistic Interactions of Epigallocatechin-3-gallate with Antiangiogenic and Antitumor Compounds Author: Javier A. Garc´ ıa-Vilas Ana R. Quesada Miguel ´ Angel Medina PII: S2213-7130(16)30005-0 DOI: http://dx.doi.org/doi:10.1016/j.synres.2016.05.001 Reference: SYNRES 20 To appear in: Received date: 30-11-2015 Revised date: 19-4-2016 Accepted date: 31-5-2016 Please cite this article as: Javier A.Garc´ ıa-Vilas, Ana R.Quesada, Miguel ´ Angel Medina, Screening of Synergistic Interactions of Epigallocatechin-3-gallate with Antiangiogenic and Antitumor Compounds, Synergy http://dx.doi.org/10.1016/j.synres.2016.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Screening of Synergistic Interactions of Epigallocatechin-3-gallate with Antiangiogenic and Antitumor Compounds Javier A. García-Vilas, Ana R. Quesada1,2, Miguel Ángel Medina1,2* 1Universidad de Málaga, Andalucía Tech, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, e IBIMA (instituto de Biomedicina de Málaga), Málaga. 2Unidad 741, CIBER de Enfermedades Raras (CIBERER). *Corresponding author: Dr. Miguel Ángel Medina, Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, E-29071, Málaga. Phone: +34952137132. Fax. +34-952131674. E-Mail: [email protected] 2 HIGHLIGHTS  A screening of combinations with EGCG is carried out with the MTT assay.  EGCG synergistically interact with 4-methylumbelliferone.  EGCG is an antagonist of the antiproliferative effects of vitamin D3. 3 Summary Purpose: To screen for possible synergistic interactions of epigallocatechin-3-gallate (EGCG) with a selection of 10 anti-angiogenic or anti-tumor compounds on the survival of endothelial and tumor cells. Methods: Human HMEC endothelial and MDA-MB231 breast cancer cells were treated with different concentrations of EGCG and the 10 tested compounds either as single agents or in paired combinations with EGCG for 3 days and final survival of cells was determined by the MTT assay. IC50 values, sensitization factors and combination indexes were calculated. Results; IC50 values of 140±2 and 45±6 µM were determined for EGCG-treated endothelial and tumor cells, respectively. IC50 values for all tested compounds were within the micromolar and the submillimolar range. The values of the sensitization factor increased and those of the combination index decreased for paired combinations of EGCG with 4-methylumbelliferone. The opposite was true for the combination of EGCG with vitamin D3. Other tested combinations did not exhibit a clear monotonic effect but rather a biphasic behaviour. Conclusion: Combinations of EGCG and 4-methylumbelliferone synergistically decrease endothelial and tumor cell survival. In contrast, the presence of EGCG antagonizes with the antiproliferative effect exerted by vitamin D3 on endothelial and tumor cells. Keywords: EGCG; angiogenesis; cancer; synergy; MTT assay 4 1. Introduction Solid cancers have several common characteristics that Hanahan & Weinberg named as the hallmarks of cancer [1]. Angiogenesis is an essential hallmark of cancer because tumor cells need oxygen and nutrients delivered by the vascular system [2]. In fact, tumor growth and metastasis are angiogenesis dependent processes, and microvascular endothelial cells recruited by tumors have become an important target in cancer therapy [1-3]. Drug discovery efforts have identified several potential therapeutic targets in endothelial cells and selective inhibitors capable of slowing tumor growth or producing tumor regression by blocking angiogenesis in in vivo tumor models [4]. However, the currently available therapies have limited success in patients, due to complex mechanisms of resistance of the tumor cells [5,6]. One approach to overcoming these problems is to use combinations of drugs with different modes of action that may lead to enhanced antitumor and antiangiogenic effects without injuring the host [5-7]. The combined use of two drugs may sometimes produce enhanced, unchanged or diminished effects in comparison with their individual effects. These three different types of behaviour of the interacting drugs are called synergy, additive/indifferent and antagonistic effects [8]. Our research group is devoted to the screening, identification and characterization of new modulators of angiogenesis. In the last years we have identified a number of natural compounds as new inhibitors of angiogenesis [see for instance 9-14]. We have also claimed for the need of combinatorial approaches to manage angiogenesis [5]. Epigallocatechin-3-gallate (EGCG) is an abundant polyphenol in green tea leaves with a number of biological activities, including antiangiogenesis [5], anticarcinogenesis [16- 5 18], antimetastasis [19], as well as cancer chemoprevention, among others [20]. Furthermore, our group has previously shown that EGCG is a potent antiinflammatory compound [21,22] targeting histidine decarboxylase [23-25]. In the present work, we analyze paired combinations of EGCG with other 10 bioactive compounds (see Table 1), some described by our research group as potent antiangiogenic compounds, and others currently used in clinical therapy [11-14, 26-32]. 2. Material and methods 2.1. Materials Metformin, leflunomide, TNP-470, sunitinib malate, kahweol, cholecalciferol (vitamin D3), 4-methylumbelliferone, dimethyl fumarate, Paclitaxel and epigallocatechin-3gallate were purchased from Sigma Chemicals Co. (St. Louis MO, USA). Hydroxytyrosol was supplied by Extrasynthèse (Lyon, France). Supplements and other chemicals not listed in this section were obtained from Sigma Chemicals Co. (St. Louis MO, USA). Cell culture media, penicillin, streptomycin and amphotericin B were purchased from Biowhittaker (Walkersville, MD, USA). Fetal bovine serum (FBS) and human serum (HS) were products of Harlan-Seralab (Belton, United Kindom). Plastics for cell culture were supplied by NUNC (Roskilde, Denmark) and VWR (West Chester, Pennsylvania, USA). 2.2. Cell culture The immortalized human microvascular endothelial cell (HMEC) line was kindly supplied by Dr. Arjan W. Griffioen (Maastrich University, The Netherlands). This immortalized cell line has been previously characterized [33]. HMEC cells were grown 6 in RPMI 1640 medium supplemented with glutamine (2mM), penicillin (50 IU/mL), streptomycin (0.05 mg/mL), and amphotericin (1.25 mg/L) supplemented with 10% fetal bovine serum, and 10% human serum. Human breast cancer carcinoma MDA-MB231 cells were purchased from ATCC and maintained in RPMI 1640 supplemented with glutamine (2mM), penicillin (50 IU/mL), streptomycin (0.05 mg/mL), and amphotericin (1.25 mg/L) supplemented with 10% fetal bovine serum. Cell cultures were maintained at 37°C under a humidified 5% CO2 atmosphere. 2.3. MTT cell growth assay The 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) dye reduction assay in 96-well microplates was used. The assay is dependent on the reduction of MTT by mitochondrial dehydrogenases of viable cells to a blue formazan product, which can be measured spectrophotometrically. Tumor cells (2.5x103 cells in a total volumen of 100 μL of complete medium) were incubated in each well with serial dilutions of single compounds or with paired combinations. After 3 days of incubation in the dark (37°C, 5% CO2 in a humid atmosphere), 10 μL of MTT (5mg/mL in PBS) was added to each well, and the plate was incubated for a further 4 h (37°C). The formazan was solved in 150 μM of 0.04 N HCl-2 propanol, and samples were spectrophotometrically measured at 550 nm. All determinations were carried out in quadruplicate, and at least three independent experiments were carried out. IC50 values were calculated as those concentrations of compound yielding 50% of cell survival, taking the values obtained for control as 100%. Firstly, we calculated IC50 values for each single compound on both cell lines. Once we knew these values for single compounds, we performed the different combinations of EGCG with the 10 selected compounds (Table 1) at concentrations from 4xIC50 to 1/8 IC50. 7 From these experiments, the effect of paired combinations was evaluated with two different approaches: determination of combination indexes and sensitization factors (see sections 2.4 and 2.5). 2.4. Combination index (CI) The so-called combination index (CI) is defined as: CI = [Ca/ICa] + [Cb/ICb],where Ca an Cb are the concentrations of compounds A and B used together to achieve a fixed effect. ICa and ICb are the concentrations of A and B, respectively required individually to achieve the same effect [8]. Here, we have used as a fixed effect that corresponding to IC50. A CI value of less than, equal to, and more than one, indicates synergy, additivity and antagonism, respectively, between these two compounds. 2.5. Sensitization factors The sensitization factors indicate he degree of sensitization induced by EGCG on the effects of the 10 selected compounds on cell survival. They were calculated as the ratio of IC50 (control cells)/ IC50 (EGCG treated cells). 3. Results 3.1. Studies of cell growth and survival with single compounds Figure 1 shows the survival curves (determined with the MTT assay) of both HMEC endothelial and MDA-MB231 tumor cells treated with EGCG. From these survival curves IC50 values of 140±2 and 45±6 µM were determined for endothelial and tumor cells, respectively. Following the same procedure, the dose-response histograms 14 References [1] Hanahan D, Weiberg R. Hallmarks of cancer: the next generation. Cell 2011;144:646-674. [2] Hanahan D, Weinber R. The hallmarks of cancer. Cell 2000;100:57-70. 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Evid Based Complement Alternat Med 2013;2013:185158. 19 [42] Piccioni F, Fiore E, Bayo J, Atorrasagasti C, Peixoto E, Rizzo M, Malvicini M, Tirado-González I, García MG, Alaniz L, Mazzolini G. 4-Methylumbelliferone inhibits hepatocellular carcinoma growth by decreasing IL-6 production and angiogenesis. Glycobiol 2015;25:825-835. [43] Yates TJ, López LE, Lokeshwar SD, Ortiz N, Kallifatidis G, Jordan A, Hoye K, Altman N, Lokeshwar VB. Dietary supplement 4-methylumbelliferone: an effective chemopreventive and therapeutic agent for prostate cancer. J Natl Cancer Inst 2015;107:djv085. 20 Figure legends Figure 1 Dose-response curves showing the effect of EGCG treatment on HMEC endothelial (circles) and MDA-MB231 tumor (squares) cell survival. Data are means±SD for three independent experiments (each with 4 replicates of each test point). Figure 2 HMEC endothelial cell dose-response histograms showing the effect of paired compounds. A) 4-MU/EGCG, B) DMF/EGCG, C) HT/EGCG, D) Kahweol/EGCG, E) Leflunomide/EGCG, F) Metformin/EGCG, G) Paclitaxel/EGCG, H) Sunitinib/EGCG, I) TNP-470/EGCG, J) Vit. D3/EGCG. Data are shown as means±S.D. for 3 independent experiments (each with 4 replicates of each test point). Figure 3 MDA-MB231 tumor cell dose-response histograms showing the effect of paired compounds. A) 4-MU/EGCG, B) DMF/EGCG, C) HT/EGCG, D) Kahweol/EGCG, E) Leflunomide/EGCG, F) Metformin/EGCG, G) Paclitaxel/EGCG, H) Sunitinib/EGCG, I) TNP-470/EGCG, J) Vit. D3/EGCG. Data are shown as means±S.D. for 3 independent experiments (each with 4 replicates of each test point). 21 Tables This manuscript includes 4 Tables. Compound Molecular structure Describe as… Molecular target 4-methylumbelliferone (4MU) Anti-angiogenic Inhibitor of hyaluronic acid synthesis Cholecalciferol (Vit D3) Anti-tumoral Inhibitor signalling of Wnt pathway Dimethylfumarate (DMF) Anti-angiogenic Inhibitor of VEGFR-2 Epigallocatechin gallate (EGCG) Anti-angiogenic Inhibitor of topoisomerases Hydroxytyrosol (HT) Anti-angiogenic Inhibitor of MMP2 activity Kahweol Anti-angiogenic Inhibitor of MMP2, uPA activity and VEGF Leflunomide Anti-angiogenic Inhibitor of polymerization of β-tubulin Metformin Anti-tumoral Inhibitor of TOR pathway Paclitaxel Anti-tumoral Inhibitor of the polymerization of β-tubulin Sunitinib Anti-tumoral Inhibitor of tyrosine kinases TNP-470 Anti-angiogenic Stimulating the production of cyclindependent kinase inhibitor p21 and inactive methionine aminopeptidase-2 Table 1. Names, structures and molecular targets of the compounds tested in this study. 23 Table 2. Effect of exposure schedule of EGCG on the 10 tested compounds and sensitization on HMEC endothelial cells. IC50 values are given as means±SD of three independent experiments. Table 3. Effect of exposure schedule of EGCG on the 10 tested compounds and sensitization on MDA-MB231 tumor cells. IC50 values are given as means±SD of three independent experiments. 4-MU DMF HT Kahweol Leflunomide EGCG (μM) IC50 (μM) Sensitization factor IC50 (μM) Sensitization factor IC50 (μM) Sensitization factor IC50 (μM) Sensitization factor IC50 (μM) Sensitization factor 0 626.7 ± 204.9 - 66.0 ± 2.4 - 78.6 ± 8.5 - 37.2 ± 2.9 - 210.8 ± 8.7 - 5.6 876.9 ± 2.9 0.7 88.1 ± 5.8 0.7 208.0 ± 3.9 0.4 > 148.6 < 0.2 87.4 ± 40.1 2.4 11.3 946.9 ± 146.6 0.7 60.3 ± 19.2 1.1 222.0 ± 2.5 0.4 74.4 ± 3.8 0.4 225.1 ± 70.6 0.9 22.5 < 78.3 > 8.0 50.5 ± 7.9 1.3 193.1 ± 6.5 0.4 < 4.6 > 8.1 290.2 ± 47.7 0.7 45 < 78.3 > 8.0 10.8 ± 3.6 6.1 10.1 ± 1.6 7.8 < 4.6 > 8.1 250.1 ± 15.3 0.8 90 < 78.3 > 8.0 < 8.3 > 7.0 < 9.8 > 8.0 < 4.6 > 8.1 223.0 ± 18.2 0.9 180 < 78.3 > 8.0 < 8.3 > 7.0 < 9.8 > 8.0 < 4.6 > 8.1 55.3 ± 11.7 3.8 Metformin Paclitaxel Sunitinib TNP-470 Vit. D3 EGCG (μM) IC50 (μM) Sensitization factor IC50 (μM) Sensitization factor IC50 (μM) Sensitization factor IC50 (μM) Sensitization factor IC50 (μM) Sensitization factor 0 186.8 ± 8.7 - 1.1 ± 0.2 - 10.5 ± 1.2 - 8.5 ± 1.3 - 33.7 ± 3.0 - 5.6 > 747.0 < 0.2 > 4.6 < 0.2 8.1 ± 2.6 1.3 15.0 ± 7.2 0.6 > 174.8 < 0.2 11.3 > 747.0 < 0.2 > 4.6 < 0.2 13.8 ± 4.3 0.8 12.0 ± 3.1 0.7 > 174.8 < 0.2 22.5 > 747.0 < 0.2 > 4.6 < 0.2 6.7 ± 0.8 1.5 7.4 ± 3.3 1.1 > 174.8 < 0.2 45 > 747.0 < 0.2 2.2 ± 1.9 0.5 < 1.3 > 8.1 < 1.1 > 7.7 > 174.8 < 0.2 90 58.0 ± 3.2 3.2 < 0.1 > 11 < 1.3 > 8.1 < 1.1 > 7.7 > 174.8 < 0.2 180 < 23.3 > 8.0 < 0.1 > 11 < 1.3 > 8.1 < 1.1 > 7.7 > 5.5 < 6.1 24 Table 4. Combination index (CI) at IC50 of EGCG combined with each of the 10 tested compounds in this study. Mean values are provided. 4-MU DMF HT Kahweol Leflunomide Metformin Placlitaxel Sunitinib TNP-470 Vit D3 EGCG (HMEC) 0.75 1.13 2.00 2.71 2.00 3.0 0.77 1.50 3.60 2.30 EGCG (MDA-MB231) 0.88 2.45 1.13 2.13 1.25 1.25 2.55 1.38 1.11 > 4